Transglutaminase Type 1 and Its Cross-linking Activity Are Concentrated at Adherens Junctions in Simple Epithelial Cells*

Transglutaminase type 1 was identified as a tyrosine-phosphorylated protein from the isolated junctional fraction of the mouse liver. This enzyme was reported to be involved in the covalent cross-linking of proteins in keratinocytes, but its expression and activity in other cell types have not been examined. Northern blotting revealed that transglutaminase type 1 was expressed in large amounts in epithelial tissues (lung, liver, and kidney), which was also confirmed by immunoblotting with antibodies raised against mouse recombinant protein. Immunoblotting of the isolated junctional fraction revealed that transglutaminase type 1 was concentrated in the fraction not only as a 97-kDa form but also as forms of various molecular masses cross-linked to other proteins. In agreement with this finding, endogenous transglutaminase type 1 was immunofluorescently colocalized with E-cadherin in cultured simple epithelial cells. In the liver and kidney, immunoelectron microscopy revealed that transglutaminase type 1 was concentrated, albeit not exclusively, at cadherin-based adherens junctions. Furthermore, by in vitro and in vivolabeling, transglutaminase cross-linking activity was also shown to be concentrated at intercellular junctions of simple epithelial cells. These findings suggested that the formation of covalently cross-linked multimolecular complexes by transglutaminase type 1 is an important mechanism for maintenance of the structural integrity of simple epithelial cells, especially at cadherin-based adherens junctions.

Cell-cell adhesion is essential for the formation and maintenance of the integrity of animal body as a community of a wide variety of cells (1). During development, the intercellular junctional complex, composed of tight junctions, adherens junctions (AJs), 1 and desmosomes (2), is repeatedly destroyed and formed. Therefore, the mechanism of regulation of the forma-tion and destruction of the junctional complex has attracted a great deal of interest from cell as well as developmental biologists. AJs are electron microscopically characterized by their electron-dense plasmalemmal undercoats through which actin filaments are densely associated with plasma membranes (3,4). E-cadherin (5), a transmembrane protein responsible for Ca 2ϩ -dependent cell-cell adhesion, is concentrated and functions as a major adhesion molecule at AJs (6). Furthermore, several constituents of AJ undercoats, which may connect Ecadherin to the actin-based cytoskeleton or regulate some AJ functions, have been identified, including ␣-, ␤-, and ␥-catenins (7-10), vinculin (11), and p120 (Cas; Ref. 12). The molecular mechanism of the formation and destruction of AJs can thus be rephrased as the mechanism responsible for the assembly and disassembly of the multimolecular complexes consisting of Ecadherin and these undercoat-constitutive proteins. Tyrosine phosphorylation of proteins has been shown to be directly involved in the assembly and disassembly of AJs (13)(14)(15)(16).
We developed a procedure for the isolation of AJs ("junctional fraction") from the rat liver (17). In the past decade, this fraction has allowed the identification of several AJ-associated proteins such as ␤-catenin, tenuin, radixin, afadin, and ponsin (8, 18 -21). Several src family tyrosine kinases (c-src, c-yes, and lyn kinases) were also shown to be concentrated in this fraction as autophosphorylated forms (22). As a continuation of this study, here we attempted to identify heavily tyrosine-phosphorylated proteins in the isolated junctional fraction from the mouse liver. Unexpectedly, an enzyme with protein cross-linking activity, transglutaminase type 1 (TGase1), was identified as a tyrosine-phosphorylated form.
TGase is a general term for enzymes that catalyze the posttranslational cross-linking of proteins by acyl transfer reaction (for reviews see Refs. [23][24][25]. In this reaction, ␥-carboxamide groups of peptide-bound glutamine residues and primary amines of peptide-bound lysine serve as acyl donor and acceptor substrates, respectively, to form either ⑀-(␥-glutamyl)lysine bonds. The resulting cross-linking bonds are covalent, stable, and resistant to proteolysis, thereby increasing the resistance of proteins to chemical, enzymatic, and physical degradation. To date, at least five distinct types of TGases have been identified; four intracellular forms (TGase1-TGase4) and one extracellular form (plasma coagulation factor XIIIa). Among these, TGase2, the so-called "tissue-type" TGase, was first identified in the liver (26), and it has been suggested to be involved in cell-to-matrix adhesion (27,28), apoptosis (29), and signal transduction as a GTPase (30), but its physiological relevance is not fully understood. Next, plasma coagulation factor XIIIa was shown to be a TGase (31). TGase3 and TGase4 were identified as "epidermal-type" and "prostate-type" enzymes, respectively, because of their tissue-specific expression patterns (32,33). TGase1, the so-called "keratinocyte-type" TGase, was identified and analyzed in epidermal keratinocytes (34 -37). TGase1 is directly involved in the formation of the cornified cell envelope of terminally differentiated epidermis by cross-linking proteins beneath plasma membranes (38). Several mutations in the TGase1 gene were reported in some families with lamellar ichthyosis, an autosomal recessive skin disorder, which is evident clinically at birth as a "collodion baby" accompanied with erythroderma (39). TGase1 has been suggested to be expressed exclusively in the skin epidermis, and its expression and functions in other tissues have not been examined in detail. TGase1-deficient mice were reported to die at an early neonatal stage, but their histological changes in tissues other than the skin epidermis have not been examined in detail (40). Identification of TGase1 in the isolated junctional fraction from the liver prompted us to evaluate the intriguing possibility that TGase-dependent cross-linking of proteins plays some important role in the regulation of AJ assembly and disassembly.
Isolation of Tyrosine-phosphorylated Proteins from Mouse Isolated Junctional Fraction-The junctional fraction was prepared from the liver of 6-week-old mice through the crude membrane and the bile canaliculi fractions according to the method described previously (17). For in vitro phosphorylation, an equal amount of each fraction was suspended in phosphorylation buffer (20 mM 1,4-piperazinediethanesulfonic acid, pH 7.0, 10 mM MnCl 2 , 2 mM MgCl 2 , 0.1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, 2 mg/ml leupeptin). These samples were then incubated with 10 mM ATP for 15 min at room temperature. The phosphorylation reaction was stopped by adding 0.1 volume of 10 ϫ solubilization buffer (100 mM Tris-HCl, pH 7.4, 10% SDS, 10 mM Na 3 VO 4 ) followed by boiling at 100°C for 5 min. Then the mixture was diluted 10 times with radioimmunoprecipitation assay (ϪSDS) buffer (10 mM Tris-HCl, pH 7.4, 1% Triton X-100, 1% deoxycholic acid sodium salt, 150 mM NaCl, 1 mM EDTA). After 1 h of centrifugation at 100,000 ϫ g, the supernatant was incubated with protein G-Sepharose 4B beads (Amersham Pharmacia Biotech) at 4°C for 4 h, which had been covalently coupled with anti-phosphotyrosine mAb (4G10) by dimethylpimelimidate⅐HCl (Pierce). After the beads were washed with 20 volumes of radioimmunoprecipitation assay buffer, bound phosphoproteins were eluted with an elution buffer (100 mM phenylphosphate, 10 mM Tris-HCl, pH 7.4, 0.01% Triton X-100, 150 mM NaCl). The eluate was then subjected to SDS-polyacrylamide gel electrophoresis. The bands to be examined were excised, and amino acid sequence analysis was performed by in-gel digestion (42). Several peptide peaks were sequenced for each band.
Cloning and Sequencing of Mouse TGase1 cDNA-Based on a mouse expressed sequence tag clone (GenBank accession number W42176) showing significant similarity to human TGase1, a partial cDNA fragment was obtained by reverse transcription polymerase chain reaction using mouse liver mRNA. This fragment was used as a probe to screen a mouse F9 cDNA library. Three positive clones contained the entire open reading frame of mouse TGase1.
Northern and Western Blotting-The expression of TGase1 in various mouse tissues and in cultured cells was examined by Northern blotting using a mouse multiple tissue Northern blot (CLONTECH, Palo Alto, CA) and poly-(A) ϩ RNAs obtained from cultured cells. For Western blotting, proteins were electrophoretically transferred from SDS-PAGE gels onto nitrocellulose membranes, which were then detected with a biotin-streptavidin-alkaline phosphatase system (Amersham Pharma-cia Biotech).
Expression Vectors and Transfections-Human TGase1 was tagged with T7 or hemagglutinin (HA) at the NH 2 or COOH terminus, respectively, as follows. Human TGase1 cDNA was a generous gift from Dr. K. Yoneda (Kyoto University). To construct the T7-tagged TGase1 expression vector (pEFT7hTG1), a BamHI site was introduced at the start codon of human TGase1 cDNA by polymerase chain reaction. Then it was digested with BamHI-XbaI and introduced into the expression vector pEF-BOSneo, provided by Dr. S. Nagata (Osaka University, Osaka, Japan). To construct the HA-tagged TGase1 expression vector (pEFhTG1HA), human TGase1 cDNA was ligated with HA tag vector at the end of the open reading frame in frame by blunt-end ligation, and was introduced into pEF-BOSneo. EL cells were transfected using Li-pofectAMINE Plus (Life Technologies), and we isolated several independent stable clones for each transfection experiment.
Immunofluorescence and Immunoelectron Microscopy-Immunofluorescence microscopy of cultured cells and frozen tissue sections were performed as described previously (41). They were observed using a fluorescence microscope, Axiophot photomicroscope (Carl Zeiss Inc., Thornwood, NY), or an MRC 1024 confocal fluorescence microscope (Bio-Rad) equipped with a Zeiss Axiophot photomicroscope. Immunoelectron microscopy was performed using ultrathin cryosections essentially according to the method described previously (43). Samples were examined with a 1200EX electron microscope (JEOL, Tokyo, Japan).
Assay for Transglutaminase Activity-The method for detecting the transglutaminase activity in vitro and in vivo was developed on the basis of previous reports (44,45). For in vitro assay, each fraction of the mouse liver was suspended in TGase reaction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 2 mg/ml leupeptin) in the same amount of proteins, in the presence of 5 mM CaCl 2 , 10 mM EGTA or 10 mM cystamine (Sigma). Then, 8 mM 5-(biotinamido)pentylamine (Pierce) was added to each mixture followed by incubation for 1 h at 37°C. The reaction was stopped by adding an equal volume of 2 ϫ SDS buffer and boiling at 100°C for 10 min. Samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes, and biotin-labeled proteins were detected with streptavidin-conjugated alkaline phosphatase. For in vivo assay, 5-(biotinamido)pentylamine was added into the culture medium at the final concentration of 4 mM, and cells were cultured for 3 h in the absence or presence of 1 mM cystamine. Each cell lysate was collected with 2 ϫ SDS buffer and subjected to SDS-PAGE in the same amount of proteins. Biotinylated bands were detected as described above. To visualize the subcellular distribution of the endogenous transglutaminase activity, cells plated on glass coverslips were labeled with 5-(biotinamido)pentylamine as described above and were examined by immunofluorescence microscopy with fluorescein isothiocyanate (FITC)-labeled avidin (Roche Molecular Biochemicals).

RESULTS
Identification of TGase1 as a Tyrosine-phosphorylated Protein in the Junctional Fraction from Mouse Liver-As previously reported, the tyrosine kinase activity was highly concentrated in the isolated junctional fraction from the liver (19). When the crude membrane, bile canaliculi, and junctional fractions were incubated with ATP in vitro followed by immunoblotting with anti-phosphotyrosine mAb (4G10), tyrosine-phosphorylated proteins were enriched at the junctional fraction (Fig. 1A). We then isolated these tyrosine-phosphorylated proteins in the junctional fraction using an immunoaffinity column with 4G10 (Fig. 1B). Among the various isolated proteins, three bands at ϳ150, 95, and 80 kDa (Fig. 1, a-c, respectively), which were heavily tyrosine phosphorylated in the junctional fraction, were subjected to amino acid sequencing. As shown in Fig. 1B, these bands yielded several sequences that were identical to partial amino acid sequences of mouse N-cadherin, ␤-catenin, radixin, and human TGase1. Because the former three proteins are known to be directly involved in the functions of the junctional complex, some important function in junctions was also expected for TGase1.
Mouse TGase1 cDNA and Its Expression-Using a partial cDNA fragment of mouse TGase1 as a probe for hybridization, we cloned a full-length cDNA encoding mouse TGase1 from a mouse F9 cDNA library (data are available from GenBank under the accession number AF186373). The 2.7-kb cDNA con-tained an open reading frame encoding a 686-amino acid polypeptide that showed sequence similarity to human and rat TGase1 (90.6 and 95.5% identity at the amino acid sequence level, respectively).
TGase1 was reported to be expressed only in keratinocytes (46 -48). To reexamine its expression in tissues other than the skin, we performed Northern blotting using the mouse TGase1 cDNA as a probe. As shown in Fig. 2A, a single 3.0-kb band of TGase1 mRNA was detected abundantly in tissues consisting of simple epithelial cells such as lung, liver, and kidney. Also, TGase1 was expressed in the mouse simple epithelial cell line MTD-1A but not in fibroblasts such as NIH3T3 (Fig. 2B).
Next, to examine the expression of TGase1 at the protein level, we raised mAbs and pAbs in rats and rabbits, respectively, using the GST fusion protein with mouse TGase1 produced in Escherichia coli as an antigen. One mAb (TG1F-1) and one pAb (pTG1C-1) specific for mouse TGase1 were obtained. To date, it was believed to be difficult to obtain specific antibodies for TGase1, and then the specificity of these antibodies was confirmed by immunofluorescence microscopy of the frozen sections of the skin from TGase1-deficient newborn mice (Ref. 40 and Fig. 3A). When the TGase1 gene was disrupted by homologous recombination, the TGase1 staining signals with our antibodies completely disappeared from the epidermis, indicating that TGase2 and -3 expressed in TGase1-deficient epidermis were not detected by our antibodies. In agreement with the results of Northern blotting ( Fig. 2A), on Western blotting anti-TGase1 pAb (pTG1C-1) recognized a single band of ϳ97 kDa in mouse epithelial tissues (liver, kidney, and lung; Fig. 3B). Of course, this pAb clearly recognized a 97-kDa band of TGase1 in the skin. A band with the same molecular mass was also detected in cultured mouse simple epithelial cells, MTD-1A cells, but not in fibroblasts (Fig. 3C). Interestingly, on Western blots of tissues and cells expressing TGase1, both TGase1-specific mAbs and pAbs reproducibly showed a smear staining pattern in the higher molecular mass region, even in the stacking gel region, in addition to the clear intense staining of the 97-kDa band. Because TGase itself was reported to be covalently cross-linked to various proteins by its own enzy-matic activity (49 -51), this smear staining with anti-TGase1 antibodies would be attributed to the cross-linked TGase1 within cells. When TGase1 was overexpressed in cultured EL cells (Fig. 3C, EL/T7-hTG1 and EL/hTG1-HA), the intensity of the smear staining was markedly increased.
Concentration of TGase1 in Cell-to-Cell Adherens Junctions in Simple Epithelial Cells-The subcellular distribution of TGase1 in simple epithelial cells was then examined. First, liver fractions with the same amount of total proteins were subjected to Western blotting with anti-TGase1 pAb (Fig. 3B).  1. Identification of TGase1 as a tyrosine-phosphorylated protein in the junctional fraction from mouse liver. A, concentration of endogenous tyrosine kinase activity in the isolated junctional fraction from mouse liver. The crude membrane (Membrane), bile canaliculi (BC), and junctional fractions (Jun) were incubated with 10 mM ATP at 37°C for 1 h (ATP(ϩ)). As a control, fractions were incubated in the absence of ATP (ATP(Ϫ)). Each fraction was then subjected to SDS-PAGE, followed by Western blotting with anti-phosphotyrosine mAb (4G10). Note that the amounts of tyrosine-phosphorylated proteins were increased markedly in the ATP-incubated junctional fraction. B, isolation of tyrosine-phosphorylated proteins from the junctional fraction. Tyrosine-phosphorylated proteins in the ATP-incubated junctional fraction were isolated using an anti-phosphotyrosine mAb immunoaffinity column. The eluate was separated by SDS-PAGE followed by silver staining. Three heavily phosphorylated bands shown in A with molecular masses of 150, 95, and 80 kDa (a-c, respectively) were subjected to direct amino acid sequencing. Sequences from bands a and c were identical to the partial sequences of mouse N-cadherin and radixin, respectively, and two sequences obtained from band b were identical to the partial sequences of mouse ␤-catenin and human TGase1.
The density of the pAb-positive 97-kDa band showed a clear increase in the junctional fraction, indicating that TGase1 itself was concentrated in junctions as a non-cross-linked form. Furthermore, the intensity of the smear staining was also markedly increased in the higher molecular mass region as well as in the stacking gel. This finding indicated that fairly large amounts of TGase1 were concentrated in junctions where they were covalently cross-linked to various proteins to form large multimolecular complexes.
Next, the subcellular localization of TGase1 was examined in cultured mouse simple epithelial cells, Eph4 cells, by confocal immunofluorescence microscopy. As shown in Fig. 4, TGase1 and E-cadherin were diffusely co-distributed on lateral membranes with significant concentration at junctional regions (AJs). In contrast, ZO-1 was concentrated more apically than TGase1 and E-cadherin.
Immunofluorescence localization of TGase1 in epithelial tissues was difficult, partly because diffuse signals were detected with anti-TGase1 antibodies in non-junctional plasma membranes such as microvilli (data not shown), and partly because endothelial cells gave intense TGase1 signals. In frozen sections of the liver, significant staining was detected around the ZO-1-positive junctional complex regions by immunofluorescence microscopy with anti-TGase1 mAb (Fig. 5, a and b). Immunoelectron microscopy of ultrathin cryosections of liver and kidney epithelial cells revealed that TGase1 was concentrated at adherens junctions, although this was not exclusive, and not at tight junctions (Fig. 5, c, d, f, and g). TGase1 signals were also found in endothelial cells at the electron microscopic level (Fig. 5e).

Concentration of the TGase Cross-linking Activity at Cell-Cell Junctions in Simple Epithelial Cells-A method for labeling
TGase-specific substrates with the biotin-labeled primary amine 5-(biotinamido)pentylamine was reported previously (44,45). We first used this method to detect TGase activity in liver fractions from mouse. Each fraction with the same amount of total proteins was incubated with 8 mM 5-(biotinamido)pentylamine, electrophoresed, and transferred onto nitrocellulose membranes, and then the cross-linked products were detected using the avidin-alkaline phosphatase detection kit. As shown in Fig. 6A, the endogenous TGase activity, i.e. the amount of biotin-labeled proteins, was significantly concentrated in the junctional fraction. This reaction was actually

FIG. 3. Western blotting analysis of mouse TGase1 expression.
A, specificity of anti-TGase1 antibodies. Frozen sections of the skin from wild-type (a) and TGase1-deficient newborn mice (b) were stained with anti-TGase1 mAb (TG1F-1). In wild-type mice, this mAb stained the epidermis (arrows), whereas in TGase1-deficient mice the TGase1 signal became undetectable, confirming the specificity of mAb TG1F-1 for TGase1. Asterisk, dermis. Anti-TGase1 pAb (pTG1C-1) showed the same result (data not shown). B, total lysates of the mouse liver (Total) and its fractions of the cytosol (Cytosol), crude membranes (Membrane), bile canaliculi (BC), and isolated junctions (Jun) as well as total lysates of various mouse tissues were subjected to SDS-PAGE in the same amount of total proteins, followed by Western blotting with anti-TGase1 pAb (pTG1C-1). TGase1 was detected as a single 97-kDa band (large arrow) in the liver (Total), kidneys, lungs, and skin. This 97-kDa form of TGase1 was concentrated in the junctional fraction (Jun). A faint signal at ϳ150 kDa (large arrowhead) in almost all tissue lysate was nonspecifically stained because of second and third antibodies in the biotin-streptavidin-alkaline phosphatase system. C, total lysates of cultured mouse cells and EL cells expressing T7-or HA-tagged human TGase1 (EL/T7-hTG1 and EL/hTG1-HA, respectively) were subjected to SDS-PAGE in the same amount of total proteins, followed by Western blotting with anti-TGase1 pAb (pTG1C-1). The 97-kDa form of TGase1 (large arrow) was detected in simple epithelial cells but not in fibroblasts. Tagged human TGase1 molecules were detected as bands with the expected molecular masses in EL/T7-hTG1 and EL/hTG1-HA cells. Note that in the tissues and cells expressing TGase1, intense smear staining was detected in the higher molecular mass region, even in the stacking gel in B and C (between small arrowhead and small arrow).

FIG. 4. Subcellular distribution of TGase1 in cultured mouse epithelial cells. Eph4 cells were doubly stained with anti-TGase1 mAb (a and aЈ) and anti-ZO-1 pAb (b and bЈ) or anti-TGase1 mAb (d and dЈ)
and anti-E-cadherin pAb (e and eЈ) and observed by confocal immunofluorescence microscopy. All sectional images for each cell layer (30 images) were superimposed into a single image (a, b, d, and e), and then corresponding cross-sectional views were generated (aЈ, bЈ, dЈ, and eЈ). Composite images (c, f, cЈ, and fЈ) revealed that TGase1 and E-cadherin were diffusely co-distributed on lateral membranes (dЈ-fЈ and arrowheads) with significant concentration at junctional regions (arrows), and that TGase1 was concentrated more basally than ZO-1 in junctional regions (aЈ-cЈ and arrows). Scale bar, 10 m. dependent on the endogenous TGase activity, because it was completely suppressed by 10 mM EDTA or 10 mM cystamine (data not shown), potent inhibitors of TGases.
When cultured Eph4 cells were incubated with 5-(biotinamido)pentylamine, various proteins were cross-linked with this reagent by endogenous TGase activity (Fig. 6B). This labeling was completely inhibited by 1 mM cystamine. When the localization of the cross-linked proteins was examined with avidin-FITC by confocal immunofluorescence microscopy, the FITC signal overlapped with that from endogenous TGase1 at cellcell borders (Fig. 7, a-c). This signal was abolished in the absence of 5-(biotinamido)pentylamine or in the presence of cystamine in the culture medium, indicating that the FITC signals represent the cross-linking activity of endogenous TGase (Fig. 7, d-f).

DISCUSSION
TGase1 was reported to be expressed specifically in keratinocytes, and its cross-linking activity was thought to play a central role in the formation of the cornified cell envelope of terminally differentiated epidermis (38,40). In this study, however, Northern as well as Western blotting analyses indicated that TGase1 was also expressed in large amounts in tissues containing simple epithelia, such as the lungs, liver, and kidneys. Furthermore, immunofluorescence and immunoelectron microscopy revealed that endogenous TGase1 was mostly colocalized with E-cadherin and concentrated, although this localization was not exclusive, at AJs in simple epithelial cells. These findings prompted us to reexamine the physiological relevance of TGase1.
The TGase activity detected using 5-(biotinamido)pentylamine as a substrate was also enriched in the isolated junc-tional fraction from the liver and co-concentrated with E-cadherin at AJs in cultured simple epithelial cells. However, this pentylamine is not a specific substrate for TGase1. Among the four types of intracellular TGases (types 1-4), only type 2 (TGase2) was reported to be expressed in various types of cells (52). In agreement with previous reports (53), Western blotting with anti-TGase2 mAb (CUB7402; NeoMarkers, Fremont, CA) revealed that TGase2 was abundant in the cytoplasm of the liver and was not concentrated in the junctional fraction in contrast to TGase1 (data not shown). Furthermore, in cultured simple epithelial cells, TGase2 was not detected by Western blotting or immunofluorescence staining with anti-TGase2 mAb (CUB7402; data not shown). Therefore, the TGase activity detected with 5-(biotinamido)pentylamine in this study was mostly attributed to TGase1, although the possibility cannot be ruled out that as yet unidentified type of TGase gives rise to part of observed activity, and the TGase1 concentrated at Ecadherin-based cell adhesion sites appeared to be active as a transglutaminase within cells. The "membrane-associated TGase activity," which was previously reported in the liver (54), would be the same as the activity described in this study.
These findings suggested that the TGase1-mediated covalent cross-linking of proteins is directly involved in the formation and maintenance of intercellular junctions, especially AJs. To date, only the noncovalent protein-protein binding in the junctions has been intensively analyzed. Western blotting of the junctional fraction with anti-TGase1 pAb revealed that TGase1 was concentrated in the junctions not only as a full-length 97-kDa form but also as higher molecular mass forms (see Fig.  3B). Because TGase itself was known to be cross-linked to various proteins through its own enzymatic activity, in the junctions this "auto-cross-linking" also would produce higher molecular mass forms of TGase1. Furthermore, the numerous biotin-labeled bands shown in Fig. 6 indicated that in the junctions TGase1 used various proteins as substrates. Thus, in the intercellular junctions, especially in AJs, we would expect the presence of highly complicated multimolecular complexes, in which constituents including TGase1 itself were covalently cross-linked through the TGase1 enzymatic activity. Because these complexes would be resistant to extraction even with SDS, it would be difficult to analyze them with conventional biochemical methods, including SDS-PAGE.
To understand the physiological functions of TGase1 in simple epithelial cells, several important questions must be answered in future studies. The first important question is how TGase1 is targeted to the intercellular junctions, especially to AJs, in simple epithelial cells. TGase1, but not the other TGases, was reported to be acylated with myristates and/or palmitates at its NH 2 -terminal cysteine-rich region, and this was thought to be the reason why only the TGase1 was anchored on membranes. However, to be recruited to AJs, other additional mechanisms are required. Furthermore, it should be emphasized here that even in simple epithelial cells TGase1 was not exclusively distributed at AJs. Taking into consideration that TGase1 forms large multimolecular complexes that include TGase1 itself, the mechanism responsible for localization of TGase1 in simple epithelial cells would be rather complicated.
The second point to be elucidated is the identities of the major substrates for TGase1 in AJs. As discussed above, in the 5-(biotinamido)pentylamine cross-linking experiments with the isolated junctional fraction, various proteins appeared to be cross-linked by endogenous TGase1 activity. It is technically possible to isolate biotinylated, glutamine-containing proteins from the fraction using avidin-coupled columns and to determine what species of proteins are cross-linked with 5-(biotin- amido)pentylamine. Identification of the substrates of the enzymatic reaction would greatly help us analyze the cellular function of TGase1 in future studies. Third, it is necessary to determine the mechanism of regulation of TGase1 activity. We initially identified TGase1 as a tyrosine-phosphorylated protein from the junctional fraction of the mouse liver, suggesting that the TGase1 activity is regulated through its tyrosine phosphorylation. Although it was confirmed that some fraction of TGase1 was actually tyrosine phosphorylated in cultured simple epithelial cells by immunoprecipitation (data not shown), the effect of tyrosine phosphorylation on the enzymatic activity of TGase1 remained to be elucidated.
The results of this study shed light on the possible functions of TGase1 in AJs in simple epithelial cells. TGase1-deficient mice were reported to die within 4 -5 h after birth (40). It was speculated that mice died of dehydration caused by skin barrier dysfunction, because the widespread expression of TGase1 in tissues other than the skin had not been demonstrated. However, they noticed that TGase1-deficient mice were smaller than controls, and preliminary studies identified some histological changes at least in the lung. Further extensive analyses of the TGase1-deficient mice will lead us to a better understanding of the physiological roles of TGase1 in simple epithelia. Because it is widely thought that the plasmalemmal undercoat structures in AJs are involved in the stabilization and/or up-regulation of cadherin-based cell adhesion, it is reasonable to speculate that TGase1-mediated cross-linking of proteins plays a role in further stabilization and up-regulation FIG. 6. Transglutaminase activity monitored by 5-(biotinamido)pentylamine labeling. A, Each fraction of the mouse liver was incubated with 8 mM 5-(biotinamido)pentylamine in the presence of 5 mM CaCl 2 or 10 mM EDTA at 37°C for 1 h. They were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and detected with the avidin-alkaline phosphatase system. The cross-linked proteins were enriched at the junctional fraction in the presence of CaCl 2 , but not in the presence of EDTA (a potent inhibitor of TGase activity), indicating that the endogenous TGase activity itself was concentrated in the junctional fraction. B, Eph4 cells were cultured in medium containing 4 mM 5-(biotinamido)pentylamine for 3 h in the absence or presence of cystamine (a potent inhibitor of TGase), and then the cross-linked proteins were analyzed as in A. Biotinylated products were detected only in the absence of cystamine at higher molecular mass regions as well as in the stacking gel (between arrowhead and arrow).
of cell-cell adhesion. However, this cross-linking is not necessarily irreversible in vivo. The enzymatic activity that catalyzes the breakdown of ␥-glutamylamines to free amines and 5-oxo-proline, i.e. the breakdown of the TGase-mediated covalent bonds, was identified in rabbit tissues such as the kidneys, liver, and intestine (55). Recently, TGase2 and factor XIIIa themselves were reported to possess such hydrolytic activities (56). Therefore, it is possible that the TGase1-mediated covalent cross-linking is dynamically regulated in simple epithelial cells. Further studies are required to characterize the physiological functions of TGase1 in epithelial cells and to determine whether the TGase1-mediated cross-linking of proteins is one of the important and general mechanisms involved in formation, maintenance, and regulation of the structural integrity of simple epithelial cells, especially at AJs.